Methods for using the obese gene and its gene product to identify hematopoietic progenitor cells

ABSTRACT

The present invention relates to methods for using various forms of a novel receptor expressed by hematopoietic and endothelial cells. An additional variant form of this receptor has been detected in brain cells and shown to bind to the obese gene product, leptin. Therefore, leptin may be used to stimulate the growth and development of receptor-positive hematopoietic and endothelial cells in vitro and in vivo. In addition, this receptor is selectively expressed in hematopoietic progenitor cells with long-term repopulating potential. Thus, agents that specifically bind to this receptor may be used to identify and isolate progenitor cells for a variety of clinical applications.

The present application is a continuation of co-pending U.S. patentapplication Ser. No. 08/618,957, filed Mar. 20, 1996, which is acontinuation-in-part of U.S. patent application Ser. No. 08/589,915,filed Jan. 23, 1996, (now abandoned) which is a continuation-in-part ofU.S. patent application Ser. No. 08/355,888, filed Dec. 14, 1994 (nowU.S. Pat. No. 5,763,211), which is a continuation-in-part of U.S. patentapplication Ser. No. 08/306,231 filed Sep. 14, 1994 (now U.S. Pat. No.5,643,748), each of which is incorporated by reference herein in itsentirety.

1. INTRODUCTION

The present invention relates to methods for using various forms of anovel receptor expressed by hematopoietic and endothelial cells. Anadditional variant form of this receptor has been detected in braincells and shown to bind to the obese gene product, leptin. Therefore,leptin may be used to stimulate the growth and development ofreceptor-positive hematopoietic and endothelial cells in vitro and invivo. In addition, this receptor is selectively expressed inhematopoietic progenitor cells with long-term repopulating potential.Thus, agents that specifically bind to this receptor may be used toidentify and isolate progenitor cells for a variety of clinicalapplications.

2. BACKGROUND OF THE INVENTION

2.1. Hematopoietin Receptor Gene Family

A variety of diseases, including malignancy and immunodeficiency, arerelated to malfunction within the lympho-hematopoietic system. Some ofthese conditions could be alleviated and/or cured by repopulating thehematopoietic system with progenitor cells, which when triggered todifferentiate would overcome the patient's deficiency. Therefore, theability to initiate and regulate hematopoiesis is of great importance(McCune et al., 1988, Science 241: 1632).

The process of blood cell formation, by which a small number ofself-renewing stem cells give rise to lineage specific progenitor cellsthat subsequently undergo proliferation and differentiation to producethe mature circulating blood cells has been shown to be at least in partregulated by specific hormones. These hormones are collectively known ashematopoietic growth factors or cytokines (Metcalf, 1985, Science 229:16; Dexter, 1987, J. Cell Sci. 88: 1; Golde and Gasson, 1988, ScientificAmerican, July: 62; Tabbara and Robinson, 1991, Anti-Cancer Res. 11: 81;Ogawa, 1989, Environ. Health Presp. 80: 199; Dexter, 1989, Br. Med.Bull. 45: 337).

With the advent of recombinant DNA technology, the genes encoding anumber of these molecules have now been molecularly cloned and expressedin recombinant form (Souza et al., 1986, Science 232: 61; Gough et al.,1984, Nature 309: 763; Yokota et al., 1984, Proc. Natl. Acad. Sci.U.S.A. 81: 1070; Kawasaki et al., 1985, Science 230: 291). Thesecytokines have been studied in their structure, biology and eventherapeutic potential. Some of the most well characterized factorsinclude erythropoietin (EPO), stem cell factor (SCF), granulocytemacrophage colony stimulating factor (GM-CSF), macrophage colonystimulating factor (M-CSF), granulocyte colony stimulating factor(G-CSF), the interleukins (IL-1 to IL-15) and thrombopoietin (TPO).

These cytokines act on different cell types at different stages duringblood cell development, and their potential uses in medicine arefar-reaching which include blood transfusions, bone marrowtransplantation, correcting immunosuppressive disorders, cancer therapy,wound healing, and activation of the immune response (Golde and Gasson,1988, Scientific American, July: 62).

Apart from inducing proliferation and differentiation of hematopoieticprogenitor cells, such cytokines have also been shown to activate anumber of functions of mature blood cells (Stanley et al., 1976, J. Exp.Med. 143: 631; Schrader et al., 1981, Proc. Natl. Acad. Sci. U.S.A. 78:323; Moore et al., 1980, J. Immunol. 125: 1302; Kurland et al., 1979,Proc. Natl. Acad. Sci. U.S.A. 76: 2326; Handman and Burgess, 1979, J.Immunol. 122: 1134; Vadas et al., 1983, Blood 61: 1232; Vadas et al.,1983, J. Immunol. 130: 795), including influencing the migration ofmature hematopoietic cells (Weibart et al., 1986, J. Immunol. 137:3584).

Cytokines exert their effects on target cells by binding to specificcell surface receptors. A number of cytokine receptors have beenidentified and the genes encoding them molecularly cloned. Severalcytokine receptors have recently been classified into a hematopoietinreceptor (HR) superfamily. The grouping of these receptors was based onthe conservation of key amino acid motifs in the extracellular domains(Bazan, 1990, Immunology Today 11: 350). The HR family is defined bythree conserved motifs in the extracellular domain of its members. Thefirst is a Trp-Ser-X-Trp-Ser (WSXWS box) motif which is highly conservedand located amino-terminal to the transmembrane domain. Most members ofthe HR family contain this motif. The second consists of four conservedcysteine residues located in the amino-terminal half of theextracellular region. The third is that both the conserved cysteines andthe WSXWS box are found within two separate conserved fibronectin TypeIII (FN III) domains. The members of the HR family include receptors forligands such as EPO, G-CSF (Fukunaga, 1990, Cell 61: 341), GM-CSF, IL-3,IL-4, IL-5, IL-6, IL-7, IL-2 (β-subunit), IL-12, IL-13, IL-15 and LIF(Cosman, 1990, TIBS 15: 265).

Ligands for the HR are critically involved in the proliferation,maturation and differentiation of blood cells. For example, IL-3promotes the proliferation of early multilineage pluripotent stem cells,and synergizes with EPO to produce red cells. IL-6 and IL-3 synergize toinduce proliferation of early hematopoietic precursors. GM-CSF has beenshown to induce the proliferation of granulocytes as well as increasemacrophage function. IL-7 is a bone marrow-derived cytokine that plays arole in producing immature T and B lymphocytes. IL-4 inducesproliferation of antigen-primed B cells and antigen-specific T cells.Thus, members of this receptor superfamily are involved in theregulation of the hematopoietic system.

2.2. The Obese Gene, Gene Product and its Receptor

In order to study the molecular mechanism of weight regulation, Zhang etal. (1994, Nature 372: 425-432) cloned the mouse obese (ob) gene fromob/ob mice, which contain a single nucleotide mutation resulting in anobese phenotype. When an isolated gene fragment was used as a probe, itwas shown to hybridize with RNA only in white adipose tissue by northernblot analysis, but not with RNA in any other tissue. In addition, thecoding sequence of the ob gene hybridized to all vertebrate genomic DNAstested, indicating a high level of conservation of this molecule amongvertebrates. The deduced amino acid sequences are 84% identical betweenhuman and mouse, and both molecules contain features of secretedproteins.

In an effort to understand the physiologic function of the ob gene,several independent research groups produced recombinant ob gene productin bacteria for in vivo testing (Pelleymounter et al., 1995, Science269: 540-543; Halaas et al. 1995, Science 269: 543-546; Campfield etal., 1995, Science 269: 546-549). When the OB protein (also known asleptin) was injected into grossly obese mice, which possessed two mutantcopies of the ob gene, the mice exhibited a reduced appetite and beganto lose weight. More importantly, Campfield et al. (1995, Science 269:546-549) injected leptin directly into lateral ventricle, and observedthat the animals reduced their food intake, suggesting that leptin actson central neuronal networks to regulate feeding behavior and energyexpenditure. This result also provided evidence that leptin-responsivecells might reside in the brain.

Recently, a leptin fusion protein was generated and used to screen for aleptin receptor (OB-R) in a cDNA expression library prepared from mousechoroid plexus, a tissue that lines brain cavities termed ventricles(Tartaglia et al., 1995, Cell 83: 1263-1271). This approach led to thecloning of one form of the OB-R coding sequence, which reveals a singlemembrane-spanning receptor, sharing structural similarities with severalClass I cytokine receptors, such as the gp130 signal-transducingcomponent of the IL-6 receptor (Taga et al., 1989, Cell 58: 573-581),the G-CSF receptor (Fukunaga et al., 1990, Cell 61: 341-350), and theleukemia inhibitory factor receptor (Gearing et al., 1991, EMBO J. 10:2839-2848). Northern blot analysis and reverse transcription-polymerasechain reaction (RT-PCR) demonstrate that OB-R mRNA is expressed inseveral tissues, including lung, kidney, total brain and hypothalamus,but there was no report on the expression of OB-R in hematopoietictissues.

The mouse OB-R isolated by Tartaglia, et al., contains a relativelyshort intracellular cytoplasmic domain as compared with other Class Icytokine receptors. Subsequently, when its human homolog was isolatedfrom a human infant brain library, its predicted protein sequencecontains a much longer intracellular domain. In view of this finding, itwas speculated that different forms of the receptor might exist(Barinaga, 1996, Science 271: 29). However, prior to the presentinvention, no alternative forms of the OB-R had been identified.

3. SUMMARY OF THE INVENTION

The present invention relates to methods for using Hu-B1.219 (or OB-R)variants as markers for the identification and isolation of progenitorcells in the hematopoietic and endothelial lineages, and methods forusing the ob gene and its gene product, leptin, to stimulatehematopoietic and endothelial development.

The invention is based, in part, on the Applicants' discovery of threeforms of a novel member of the HR family, designated Hu-B1.219, whichhave been isolated from a human fetal liver cDNA library. Sequencecomparison of these molecules with a human OB-R sequence (Tartaglia etal., 1995, Cell 83: 1263-1271) shows that they are nearly identical intheir extracellular domains. Therefore, these four molecules representvariant forms of the receptor that respond to leptin as a ligand. Whilethe three isoforms described herein differ from the reported OB-Rprotein at only three amino acid positions in the extracellular domain,all four variants contain extensive differences in their intracellulardomains at their 3′ ends. Thus, although these receptors bind to leptin,they may transduce different signals upon ligand binding. In addition,Hu-B1.219 is expressed in several cell lines of hematopoietic andendothelial origin. Tissue expression analysis demonstrates that fetallung and liver also contain high levels of its mRNA. Moreover, humanCD34⁺ bone marrow cells express Hu-B1.219. When mouse fetal liver cellsare separated into several fractions based on their expression of AA4.1and FcR, the expression of the mouse homolog of Hu-B1.219 is detectedexclusively in the AA4.1⁺/FcR⁻ population, which has been shown tocontain most if not all of the long-term repopulating hematopoieticprogenitor cells at this stage of fetal liver development. Furthermore,mouse bone marrow cells proliferate and differentiate in response toleptin stimulation by producing erythroid colony-forming units (CFU-e),erythroid burst-forming units (BFU-e) and granulocyte-macrophage(CFU-GM) colonies. Freshly isolated murine yolk sac cells also produceerythroid growth following leptin stimulation. Additionally, Hu-B1.219is expressed in some endothelial cells and their precursors as they arederived from the embryonic yolk sac. Therefore, Hu-B1.219 is a markerfor hematopoietic and endothelial progenitor cells.

A wide variety of uses are encompassed in the present invention,including but not limited to, the use of Hu-B1.219-specific bindingagents to identify and isolate hematopoietic and endothelial progenitorcells, the use of leptin to activate such progenitor cells for in vitroor ex vivo expansion, the use of leptin for in vivo stimulation of thesame cell population in patients with immunodeficiency and anemia, andthe use of leptin to promote angiogenesis and vasculogenesis, as well asaugmentation of donor cell engraftment following bone marrowtransplantation.

4. BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-1G Nucleotide sequence (SEQ ID NO: 1) and deduced amino acidsequences (SEQ ID NOs: 2, 3, & 4) of Form 1 of Hu-B1.219. The 3′ end ofthis molecule is 98% identical to a human retrotransposon sequence.

FIG. 2. Nucleotide Sequence comparison between Hu-B1.219 Form 1 (SEQ IDNO: 5), Form 2 (SEQ ID NO: 6) and Form 3 (SEQ ID NO: 7) at the 3′ end.

FIG. 3A-3F Amino acid comparison between Hu-B1.219 Forms 1(HuB1.219-1)(SEQ ID NO: 8), 2 (HuB1.219-2)(SEQ ID NO: 9), 3(HuB1.219-3)(SEQ ID NO: 10), human OB-R (HuOBR)(SEQ ID NO: 11) andmurine OB-R (MuOBR)(SEQ ID NO: 12).

FIG. 4. Human adult multiple tissue northern blots are carried out witha probe derived from the extracellular domain of Hu-B1.219 according tothe manufacturer's instructions (Clontech, Palo Alto, Calif.).

FIGS. 5A and 5B PCR analysis of Hu-B1.219 expression in human CD34⁺ andCD34⁻ cells. FIG. 5A is detected with Hu-B1.219 primers, whereas FIG. 5Bis detected with CD34 primers. BMMNC represents bone marrow mononuclearcells as positive controls.

FIGS. 6A and 6B PCR analysis of murine Hu-B1.219 expression in mousefetal liver subpopulations separated by antibodies to AA4.1 and FcR.FIG. 6A is detected with Hu-B1.219 primers, whereas FIG. 6B is detectedwith CD34 primers. NS represents non-sorted cells and BM represents bonemarrow cells as controls.

FIG. 7. Age matched female mice from normal, ob/ob and db/db strainswere used as marrow donors for erythroid colony forming assayscontaining low serum concentration. □=medium containing IL-3, GM-CSF andEPO; ▪=medium containing IL-3, GM-CSF, EPO and recombinant murineleptin.

FIG. 8. Three types of bone marrow colonies are stimulated by leptin:CFU-e, BFU-e and CFU-GM. □=medium containing IL-3, GM-CSF and EPO;▪=medium containing IL-3, GM-CSF, EPO and recombinant murine leptin.

5. DETAILED DESCRIPTION OF THE INVENTION

5.1. Expression of the OB-R in Hematopoietic Cells

The present invention relates to a novel hematopoietic progenitor cellmarker and its use for cell identification and isolation, as well as theuse of leptin to stimulate hematopoietic and endothelial development viathis receptor known as Hu-B1.219. In a specific embodiment by way ofexample in Section 6, infra, several forms of this receptor were clonedand characterized. Amino acid sequence comparison of these relatedmolecules with a published human OB-R sequence (Tartaglia et al., 1995,Cell 83: 1263-1271) reveals only three amino acid differences in theirextracellular domains but extensive diversity in their intracellularcytoplasmic domains. More specifically, FIG. 1A-1G shows that in theHu-B1.219 molecules described herein, nucleotide residues #349-351encode alanine, nucleotide residues #421-423 encode arginine andnucleotide residues #763-765 encode arginine. Additionally, the fourforms diverge both in length and sequence composition from nucleotideresidue #2771 and beyond. In this regard, the intracellular domain ofForm 1 of Hu-B1.219 described herein is highly homologous to aretrotransposon sequence (Ono et al., 1987, Nucl. Acid. Res. 15:8725-8737).

Analysis of the Hu-B1.219 variants reveals significant homology to theFN III domain of the HR family indicating that they belong to the HRfamily of receptors. Northern blot hybridization and RT-PCR analysesindicates that Hu-B1.219 mRNA is highly expressed in cells ofhematopoietic and endothelial origin. In addition, the Hu-B1.219sequence is expressed in certain fetal tissues and tumor cell lines.Hence, in addition to its expression in the brain for weight regulationby leptin, Hu-B1.219 (or OB-R) is expressed by hematopoietic andendothelial cells, thereby rendering these cells responsive to theaction of leptin.

Since additional variant forms of the molecule may exist, they can beidentified by labeled DNA probes made from nucleic acid fragmentscorresponding to any portion of the cDNA disclosed herein in a cDNAlibrary prepared from human fetal liver, human lung, human kidney, humanchoroid plexus, human hypothalamus, human prostate and human ovary. Morespecifically, oligonucleotides corresponding to either the 5′ or 3′terminus of the cDNA sequence may be used to obtain longer nucleotidesequences. Briefly, the library may be plated out to yield a maximum of30,000 pfu for each 150 mm plate. Approximately 40 plates may bescreened. The plates are incubated at 37° C. until the plaques reach adiameter of 0.25 mm or are just beginning to make contact with oneanother (3-8 hours). Nylon filters are placed onto the soft top agaroseand after 60 seconds, the filters are peeled off and floated on a DNAdenaturing solution consisting of 0.4N sodium hydroxide. The filters arethen immersed in neutralizing solution consisting of 1M Tris HCL, pH7.5, before being allowed to air dry. The filters are prehybridized incasein hybridization buffer containing 10% dextran sulfate, 0.5M NaCl,50 mM Tris HCL, pH 7.5, 0.1% sodium pyrophosphate, 1% casein, 1% SDS,and denatured salmon sperm DNA at 0.5 mg/ml for 6 hours at 60° C. Theradiolabelled probe is then denatured by heating to 95° C. for 2 minutesand then added to the prehybridization solution containing the filters.The filters are hybridized at 60° C. for 16 hours. The filters are thenwashed in 1× wash mix (10× wash mix contains 3M NaCl, 0.6M Tris base,and 0.02M EDTA) twice for 5 minutes each at room temperature, then in 1×wash mix containing 1% SDS at 60° C. for 30 minutes, and finally in 0.3×wash mix containing 0.1% SDS at 60° C. for 30 minutes. The filters arethen air dried and exposed to x-ray film for autoradiography. Afterdeveloping, the film is aligned with the filters to select a positiveplaque. If a single, isolated positive plaque cannot be obtained, theagar plug containing the plaques will be removed and placed in lambdadilution buffer containing 0.1M NaCl, 0.01M magnesium sulfate, 0.035MTris HCl, pH 7.5, 0.01% gelatin. The phage may then be replated andrescreened to obtain single, well isolated positive plaques. Positiveplaques may be isolated and the cDNA clones sequenced using primersbased on the known cDNA sequence. This step may be repeated until a fulllength cDNA is obtained.

One method for identifying all 31 isoforms is to PCR amplify the 31 endsof the variant cDNA from a variety of tissues including but not limitingto, choroid plexus, hypothalamus, fetal liver, bone marrow, ovary, orprostate. To obtain the 3′ end of the cDNA, an oligo-dT primer is usedto synthesize the cDNA first strand. Hu-B1.219 specific primers from theconserved region of the gene (e.g. up stream of nucleotide 2770) andoligo-dT are then used to amplify the 3′ end. The PCR fragments arecloned and sequenced by standard techniques. Once obtained, thesesequences may be translated into amino acid sequence and examined forcertain landmarks such as continuous open reading frame, regulatoryregions that associate with tyrosine kinase activation, and finallyoverall structural similarity to known variants.

5.2. Hu-B1.219 AS A Progenitor Cell Marker

Hu-B1.219 is expressed in cells of hematopoietic and endothelial origin.In a specific embodiment by way of example in Section 7, infra,Hu-B1.219 is expressed in early progenitor cells, and in a smallpercentage of progenitors with long-term repopulating potential. Inorder to utilize Hu-B1.219 receptor as a marker for cell identificationand isolation, specific binding agents such as antibodies may begenerated to the protein.

5.2.1. Generation of Antibodies

Various procedures known in the art may be used for the production ofantibodies to epitopes of natural or recombinant Hu-B1.219 receptor.Such antibodies include but are not limited to polyclonal, monoclonal,chimeric, single chain, Fab fragments and fragments produced by an Fabexpression library. Neutralizing antibodies i.e., those which competefor the ligand binding site of the receptor are especially preferred fordiagnostics and therapeutics.

Monoclonal antibodies that bind Hu-B1.219 may be radioactively labeledallowing one to follow their location and distribution in the body afterinjection. Radioisotope tagged antibodies may be used as a non-invasivediagnostic tool for imaging de novo cells of tumors and metastases aswell as fetal tissues.

Immunotoxins may also be designed which target cytotoxic agents tospecific sites in the body. For example, high affinity Hu-B1.219specific monoclonal antibodies may be covalently complexed to bacterialor plant toxins, such as diphtheria toxin, or ricin. A general method ofpreparation of antibody/hybrid molecules may involve use ofthiol-crosslinking reagents such as SPDP, which attack the primary aminogroups on the antibody and by disulfide exchange, attach the toxin tothe antibody. The hybrid antibodies may be used to specificallyeliminate Hu-B1.219 expressing tumor cells.

For the production of antibodies, various host animals may be immunizedby injection with the Hu-B1.219 protein, fragments thereof or syntheticpeptides, including but not limited to rabbits, mice, rats, hamstersetc. Various adjuvants may be used to increase the immunologicalresponse, depending on the host species, including but not limited toFreund's (complete and incomplete), mineral gels such as aluminumhydroxide, surface active substances such as lysolecithin, pluronicpolyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin,dinitrophenol, and potentially useful human adjuvants such as BCG(bacilli Calmette-Guerin) and Corynebacterium parvum.

Monoclonal antibodies to Hu-B1.219 may be prepared by using anytechnique which provides for the production of antibody molecules bycontinuous cell lines in culture. These include but are not limited tothe hybridoma technique originally described by Kohler and Milstein,(Nature, 1975, 256: 495-497), the human B-cell hybridoma technique(Kosbor et al., 1983, Immunology Today, 4: 72; Cote et al., 1983, Proc.Natl. Acad. Sci., 80: 2026-2030) and the EBV-hybridoma technique (Coleet al., 1985, Monoclonal Antibodies and Cancer Therapy, Alan R. Liss,Inc., pp. 77-96). In addition, techniques developed for the productionof “chimeric antibodies” (Morrison et al., 1984, Proc. Natl. Acad. Sci.,81: 6851-6855; Neuberger et al., 1984, Nature, 312: 604-608; Takeda etal., 1985, Nature, 314: 452-454) by splicing the genes from a mouseantibody molecule of appropriate antigen specificity together with genesfrom a human antibody molecule of appropriate biological activity can beused. Alternatively, techniques described for the production of singlechain antibodies (U.S. Pat. No. 4,946,778) can be adapted to produceHu-B1.219-specific single chain antibodies.

Antibody fragments which contain specific binding sites of Hu-B1.219 maybe generated by known techniques. For example, such fragments includebut are not limited to, the F(ab′)₂ fragments which can be produced bypepsin digestion of the antibody molecule and the Fab fragments whichcan be generated by reducing the disulfide bridges of the F(ab′)₂fragments. Alternatively, Fab expression libraries may be constructed(Huse et al., 1989, Science, 246: 1275-1281) to allow rapid and easyidentification of monoclonal Fab fragments with the desired specificityto Hu-B1.219.

5.2.2. Progenitor Cell Separation

Human hematopoietic progenitor cells may be isolated using an antibodyto Hu-B1.219 protein, a leptin ligand, a leptin peptide containing thereceptor-binding domain or a leptin fusion protein using conventionalcell separation methods well known in the art. Such Hu-B1.219-specificbinding agents may be used in combination with other agents such asanti-CD34 antibodies.

Although bone marrow is the preferred cell source, other physiologicsources of hematopoietic cells may be utilized, for example, the spleen,thymus, peripheral blood, cytokine-mobilized blood, umbilical cordblood, embryonic yolk sac, or fetal liver. Bone marrow is preferablyremoved from the iliac crest, but may also be removed from other bonecavity. Bone marrow may be removed from bone cavity by various methodswell known to those skilled in the art, including flushing the bone witha mixture of physiological media, balanced salt solution, physiologicalbuffer, and other naturally occurring factors. Typically, the bonemarrow is filtered, centrifuged and resuspended.

Once a source of hematopoietic cells is obtained, hematopoieticprogenitor cells may be separated from the cell mixture by variousmethods which utilize an agent such as a specific antibody or a leptinligand which specifically binds to cell surface Hu-B1.219-encodedreceptor protein. These techniques may include, for example, flowcytometry using a fluorescence activated cell sorter (FACS) and specificfluorochromes, biotin-avidin or biotin-streptavidin separations usingbiotin conjugated to cell surface marker-specific antibodies and avidinor streptavidin bound to a solid support such as affinity column matrixor plastic surfaces, magnetic separations using antibody-coated magneticbeads, destructive separations such as antibody and complement orantibody bound to cytotoxins or radioactive isotopes.

Separation of a cell mixture via antibodies may be performed by negativeor positive selection procedures. In negative separation, antibodies areused which are specific for markers present on undesired hematopoieticcells. Cells bound by an antibody may be removed or lysed and theremaining desired mixture retained. In positive separation, antibodiesspecific for Hu-B1.219 or leptin ligand may be used. Cells bound by theantibody or leptin are separated and retained. It will be understoodthat positive and negative separations may be used substantiallysimultaneously or in a sequential manner. It will also be understoodthat the present invention encompasses any separation technique whichcan isolate cells based on the expression of Hu-B1.219 as disclosedherein. For example, a cell mixture may be separated into CD34⁺ andCD34⁻ fractions first followed by Hu-B1.219-specific separation.

At present, the most common technique for antibody-based separation hasbeen the use of flow cytometry such as by a FACS. Typically, separationby flow cytometry is performed as follows. The suspended mixture ofhematopoietic cells are centrifuged and resuspended in media. Antibodieswhich are conjugated to fluorochrome are added to allow the binding ofthe antibodies to specific cell surface markers. The cell mixture isthen washed by one or more centrifugation and resuspension steps. Themixture is run through FACS which separates the cells based on differentfluorescence characteristics. FACS systems are available in varyinglevels of performance and ability, including multi-color analysis. Thecells can be identified by a characteristic profile of forward and sidescatter which is influenced by size and granularity, as well as bypositive and/or negative expression of certain cell surface markers.

Other separation techniques besides flow cytometry may provide forfaster separations. One such method is biotin-avidin based separation byaffinity chromatography. Typically, such a technique is performed byincubating the washed hematopoietic cells with biotin-coupled leptin orantibodies to specific markers followed by passage through an avidincolumn. Biotin-antibody-cell or biotin-leptin-cell complexes bind to thecolumn via the biotin-avidin interaction, while other cells pass throughthe column. Finally, the column-bound cells may be released byperturbation or other methods. The specificity of the biotin-avidinsystem is well suited for rapid positive separation.

Flow cytometry and biotin-avidin techniques provide highly specificmeans of cell separation. If desired, a separation may be initiated byless specific techniques which, however, can remove a large proportionof mature blood cells from the hematopoietic cell source. For example,magnetic bead separations may be used to initially remove differentiatedhematopoietic cell populations, including T-cells, B-cells, naturalkiller (NK) cells, and macrophages, as well as minor cell populationsincluding megakaryocytes, mast cells, eosinophils, and basophils.Desirably, at least about 70% and usually at least about 80% of thetotal hematopoietic cells present can be removed.

A preferred initial separation technique is density-gradient separation.Here, the bone marrow or other hematopoietic cell mixture preparation iscentrifuged and the supernatant removed. The cells are resuspended in,for example, RPMI 1640 medium (Gibco) with 10% FCS and placed in adensity gradient prepared with, for example, “FICOLL” or “PERCOLL” or“EUROCOLLINS” media. The separation may then be performed bycentrifugation or automatically with a Cobel & Cell Separator '2991(Cobev, Lakewood, Colo.). Additional separation procedures may bedesirable depending on the source of the hematopoietic cell mixture andits content. For example, if blood is used as a source of hematopoieticcells, it may be desirable to lyse red blood cells prior to theseparation of any fraction. Furthermore, elutriation may also be usedalone or in combination with all of other purification proceduresdescribed herein (Noga et al., 1990, Prog. Clin. Biol. Res. 333: 345;Noga et al., 1992, Prog. Clin. Biol. Res. 377: 411).

5.3. The Obese Gene Product, Leptin

The nucleotide and amino acid sequences of both human and mouse leptinhave been published recently by Zhang et al. (1994, Nature 372:425-432). Thereafter, the mouse coding sequence was used to expressfunctional leptin in E. coli (Pelleymounter et al., 1995, Science 269:540-543; Halaas et al., 1995, Science 269: 543-546; Campfield et al.,1995, Science 269: 546-549). Furthermore, human leptin was alsoexpressed and shown to be biologically active in murine experiments(Halaas et al., 1995, Science 269: 543-546). Hence, human, murine andhomologous coding sequences from other species may be expressed, and therecombinant protein purified by conventional techniques such as affinitychromatography with an antibody. Alternatively, natural protein may bedirectly purified from cells that secrete leptin, such as adipose celllines.

5.3.1. Expression of Leptin Protein

For the practice of the present invention, human and mouse leptinpolynucleotide sequences which encode the proteins, peptide fragments,fusion proteins or functional equivalents thereof, may be used togenerate recombinant DNA molecules that direct their expression inappropriate host cells.

Due to the inherent degeneracy of the genetic code, other DNA sequenceswhich encode substantially the same or a functionally equivalent aminoacid sequence, may be used in the practice of the invention for thecloning and expression of the leptin protein. In particular, such DNAsequences include those which are capable of hybridizing to the humanleptin sequences under stringent conditions.

Altered DNA sequences which may be used in accordance with the inventioninclude deletions, additions or substitutions of different nucleotideresidues resulting in a sequence that encodes the same or a functionallyequivalent gene product. The gene product itself may contain deletions,additions or substitutions of amino acid residues within a codingsequence, which result in a silent change thus producing a functionallyequivalent leptin protein. Such amino acid substitutions may be made onthe basis of similarity in polarity, charge, solubility, hydrophobicity,hydrophilicity, and/or the amphipathic nature of the residues involved.For example, negatively charged amino acids include aspartic acid andglutamic acid; positively charged amino acids include lysine, histidineand arginine; amino acids with uncharged polar head groups havingsimilar hydrophilicity values include the following: glycine,asparagine, glutamine, serine, threonine, tyrosine; and amino acids withnonpolar head groups include alanine, valine, isoleucine, leucine,phenylalanine, proline, methionine, tryptophan.

The DNA sequences of leptin may be engineered in order to alter thecoding sequence for a variety of ends including but not limited toalterations which modify processing and expression of the gene product.For example, mutations may be introduced using techniques which are wellknown in the art, e.g., site-directed mutagenesis, to insert newrestriction sites, to alter glycosylation patterns, phosphorylation,etc.

In another embodiment of the invention, a leptin or a modified leptinsequence may be ligated to a heterologous sequence to encode a fusionprotein. It may also be useful to encode a chimeric leptin proteinexpressing a heterologous epitope that is recognized by a commerciallyavailable antibody. A fusion protein may also be engineered to contain acleavage site located between a leptin sequence and the heterologousprotein sequence, so that the leptin protein may be cleaved away fromthe heterologous moiety.

In an alternate embodiment of the invention, the coding sequence ofleptin could be synthesized in whole or in part, using chemical methodswell known in the art. See, for example, Caruthers et al., 1980, Nuc.Acids Res. Symp. Ser. 7: 215-233; Crea and Horn, 180, Nuc. Acids Res.9(10): 2331; Matteucci and Caruthers, 1980, Tetrahedron Letters 21: 719;and Chow and Kempe, 1981, Nuc. Acids Res. 9(12): 2807-2817.Alternatively, the protein itself could be produced using chemicalmethods to synthesize leptin amino acid sequence in whole or in part.For example, peptides can be synthesized by solid phase techniques,cleaved from the resin, and purified by preparative high performanceliquid chromatography. (e.g., see Creighton, 1983, Proteins StructuresAnd Molecular Principles, W.H. Freeman and Co., N.Y. pp. 50-60). Thecomposition of the synthetic peptides may be confirmed by amino acidanalysis or sequencing (e.g., the Edman degradation procedure; seeCreighton, 1983, Proteins, Structures and Molecular Principles, W.H.Freeman and Co., N.Y., pp. 34-49).

In order to express a biologically active leptin protein, the nucleotidesequence coding for leptin, or a functional equivalent, is inserted intoan appropriate expression vector, i.e., a vector which contains thenecessary elements for the transcription and translation of the insertedcoding sequence. The leptin gene products as well as host cells or celllines transfected or transformed with recombinant leptin expressionvectors can be used for a variety of purposes.

5.3.2. Expression Systems for Leptin

Methods which are well known to those skilled in the art can be used toconstruct expression vectors containing the leptin coding sequence andappropriate transcriptional/translational control signals. These methodsinclude in vitro recombinant DNA techniques, synthetic techniques and invivo recombination/genetic recombination. See, for example, thetechniques described in Sambrook et al., 1989, Molecular Cloning ALaboratory Manual, Cold Spring Harbor Laboratory, N.Y. and Ausubel etal., 1989, Current Protocols in Molecular Biology, Greene PublishingAssociates and Wiley Interscience, N.Y.

A variety of host-expression vector systems may be utilized to expressthe leptin coding sequence. These include but are not limited tomicroorganisms such as bacteria transformed with recombinantbacteriophage DNA, plasmid DNA or cosmid DNA expression vectorscontaining the leptin coding sequence; yeast transformed withrecombinant yeast expression vectors containing the leptin codingsequence; insect cell systems infected with recombinant virus expressionvectors (e.g., baculovirus) containing the leptin coding sequence; plantcell systems infected with recombinant virus expression vectors (e.g.,cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) ortransformed with recombinant plasmid expression vectors (e.g., Tiplasmid) containing the leptin coding sequence; or animal cell systems.

The expression elements of these systems vary in their strength andspecificities. Depending on the host/vector system utilized, any of anumber of suitable transcription and translation elements, includingconstitutive and inducible promoters, may be used in the expressionvector. For example, when cloning in bacterial systems, induciblepromoters such as pL of bacteriophage λ, plac, ptrp, ptac (ptrp-lachybrid promoter) and the like may be used; when cloning in insect cellsystems, promoters such as the baculovirus polyhedrin promoter may beused; when cloning in plant cell systems, promoters derived from thegenome of plant cells (e.g., heat shock promoters; the promoter for thesmall subunit of RUBISCO; the promoter for the chlorophyll α/β bindingprotein) or from plant viruses (e.g., the 35S RNA promoter of CaMV; thecoat protein promoter of TMV) may be used; when cloning in mammaliancell systems, promoters derived from the genome of mammalian cells(e.g., metallothionein promoter) or from mammalian viruses (e.g., theadenovirus late promoter; the vaccinia virus 7.5K promoter) may be used;when generating cell lines that contain multiple copies of the leptinDNA, SV40-, BPV- and EBV-based vectors may be used with an appropriateselectable marker.

In bacterial systems a number of expression vectors may beadvantageously selected depending upon the use intended for the leptinexpressed. For example, when large quantities of leptin are to beproduced for the generation of antibodies or to screen peptidelibraries, vectors which direct the expression of high levels of fusionprotein products that are readily purified may be desirable. Suchvectors include but are not limited to the E. coli expression vectorpUR278 (Ruther et al., 1983, EMBO J. 2: 1791), in which the leptincoding sequence may be ligated into the vector in frame with the lac Zcoding region so that a hybrid AS-lac Z protein is produced; pIN vectors(Inouye & Inouye, 1985, Nucleic acids Res. 13: 3101-3109; Van Heeke &Schuster, 1989, J. Biol. Chem. 264: 5503-5509); and the like. pGEXvectors may also be used to express foreign polypeptides as fusionproteins with glutathione S-transferase (GST). In general, such fusionproteins are soluble and can easily be purified from lysed cells byadsorption to glutathione-agarose beads followed by elution in thepresence of free glutathione. The pGEX vectors are designed to includethrombin or factor Xa protease cleavage sites so that the clonedpolypeptide of interest can be released from the GST moiety.

In yeast, a number of vectors containing constitutive or induciblepromoters may be used. For a review see, Current Protocols in MolecularBiology, Vol. 2, 1988, Ed. Ausubel et al., Greene Publish. Assoc. &Wiley Interscience, Ch. 13; Grant et al., 1987, Expression and SecretionVectors for Yeast, Methods in Enzymology, Eds. Wu & Grossman, 1987,Acad. Press, N.Y., Vol. 153, pp. 516-544; Glover, 1986, DNA Cloning,Vol. II, IRL Press, Wash., D.C., Ch. 3; and Bitter, 1987, HeterologousGene Expression in Yeast, Methods in Enzymology, Eds. Berger & Kimmel,Acad. Press, N.Y., Vol. 152, pp. 673-684; and The Molecular Biology ofthe Yeast Saccharomyces, 1982, Eds. Strathern et al., Cold Spring HarborPress, Vols. I and II.

In cases where plant expression vectors are used, the expression of theleptin coding sequence may be driven by any of a number of promoters.For example, viral promoters such as the 35S RNA and 19S RNA promotersof CaMV (Brisson et al., 1984, Nature 310: 511-514), or the coat proteinpromoter of TMV (Takamatsu et al., 1987, EMBO J. 6: 307-311) may beused; alternatively, plant promoters such as the small subunit ofRUBISCO (Coruzzi et al., 1984, EMBO J. 3: 1671-1680; Broglie et al.,1984, Science 224: 838-843); or heat shock promoters, e.g., soybeanhsp17.5-E or hsp17.3-B (Gurley et al., 1986, Mol. Cell. Biol. 6:559-565) may be used. These constructs can be introduced into plantcells using Ti plasmids, Ri plasmids, plant virus vectors, direct DNAtransformation, microinjection, electroporation, etc. For reviews ofsuch techniques see, for example, Weissbach & Weissbach, 1988, Methodsfor Plant Molecular Biology, Academic Press, NY, Section VIII, pp.421-463; and Grierson & Corey, 1988, Plant Molecular Biology, 2d Ed.,Blackie, London, Ch. 7-9.

An alternative expression system which could be used to express leptinis an insect system. In one such system, Autographa californica nuclearpolyhidrosis virus (AcNPV) is used as a vector to express foreign genes.The virus grows in Spodoptera frugiperda cells. The leptin codingsequence may be cloned into non-essential regions (for example thepolyhedrin gene) of the virus and placed under control of an AcNPVpromoter (for example the polyhedrin promoter). Successful insertion ofthe leptin coding sequence will result in inactivation of the polyhedringene and production of non-occluded recombinant virus (i.e., viruslacking the proteinaceous coat coded for by the polyhedrin gene). Theserecombinant viruses are then used to infect Spodoptera frugiperda cellsin which the inserted gene is expressed. (e.g., see Smith et al., 1983,J. Virol. 46: 584; Smith, U.S. Pat. No. 4,215,051).

In mammalian host cells, a number of viral based expression systems maybe utilized. In cases where an adenovirus is used as an expressionvector, the leptin coding sequence may be ligated to an adenovirustranscription/translation control complex, e.g., the late promoter andtripartite leader sequence. This chimeric gene may then be inserted inthe adenovirus genome by in vitro or in vivo recombination. Insertion ina non-essential region of the viral genome (e.g., region E1 or E3) willresult in a recombinant virus that is viable and capable of expressingleptin in infected hosts (e.g., See Logan & Shenk, 1984, Proc. Natl.Acad. Sci. USA 81: 3655-3659). Alternatively, the vaccinia 7.5K promotermay be used. (See, e.g., Mackett et al., 1982, Proc. Natl. Acad. Sci.USA 79: 7415-7419; Mackett et al., 1984, J. Virol. 49: 857-864; Panicaliet al., 1982, Proc. Natl. Acad. Sci. USA 79: 4927-4931).

Specific initiation signals may also be required for efficienttranslation of inserted leptin coding sequences. These signals includethe ATG initiation codon and adjacent sequences. In cases where theentire leptin gene, including its own initiation codon and adjacentsequences, is inserted into the appropriate expression vector, noadditional translational control signals may be needed. However, incases where only a portion of the leptin coding sequence is inserted,exogenous translational control signals, including the ATG initiationcodon, must be provided. Furthermore, the initiation codon must be inphase with the reading frame of the leptin coding sequence to ensuretranslation of the entire insert. These exogenous translational controlsignals and initiation codons can be of a variety of origins, bothnatural and synthetic. The efficiency of expression may be enhanced bythe inclusion of appropriate transcription enhancer elements,transcription terminators, etc. (see Bittner et al., 1987, Methods inEnzymol. 153: 516-544).

In addition, a host cell strain may be chosen which modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Such modifications (e.g.,glycosylation) and processing (e.g., cleavage) of protein products maybe important for the function of the protein. The presence of severalconsensus N-glycosylation sites in leptin support the possibility thatproper modification may be important for leptin function. Different hostcells have characteristic and specific mechanisms for thepost-translational processing and modification of proteins. Appropriatecell lines or host systems can be chosen to ensure the correctmodification and processing of the foreign protein expressed. To thisend, eukaryotic host cells which possess the cellular machinery forproper processing of the primary transcript, glycosylation, andphosphorylation of the gene product may be used. Such mammalian hostcells include but are not limited to CHO, VERO, BHK, HeLa, COS, MDCK,293, WI38, etc.

For long-term, high-yield production of recombinant proteins, stableexpression is preferred. For example, cell lines which stably expressthe leptin sequence may be engineered. Rather than using expressionvectors which contain viral origins of replication, host cells can betransformed with the leptin DNA controlled by appropriate expressioncontrol elements (e.g., promoter, enhancer, sequences, transcriptionterminators, polyadenylation sites, etc.), and a selectable marker.Following the introduction of foreign DNA, engineered cells may beallowed to grow for 1-2 days in an enriched media, and then are switchedto a selective media. The selectable marker in the recombinant plasmidconfers resistance to the selection and allows cells to stably integratethe plasmid into their chromosomes and grow to form foci which in turncan be cloned and expanded into cell lines.

A number of selection systems may be used, including but not limited tothe herpes simplex virus thymidine kinase (Wigler, et al., 1977, Cell11: 223), hypoxanthine-guanine phosphoribosyltransferase (Szybalska &Szybalski, 1962, Proc. Natl. Acad. Sci. USA 48: 2026), and adeninephosphoribosyltransferase (Lowy, et al., 1980, Cell 22: 817) genes canbe employed in tk⁻, hgprt⁻ or aprt⁻ cells, respectively. Also,antimetabolite resistance can be used as the basis of selection fordhfr, which confers resistance to methotrexate (Wigler, et al., 1980,Proc. Natl. Acad. Sci. USA 77: 3567; O'Hare, et al., 1981, Proc. Natl.Acad. Sci. USA 78: 1527); gpt, which confers resistance to mycophenolicacid (Mulligan & Berg, 1981, Proc. Natl. Acad. Sci. USA 78: 2072); neo,which confers resistance to the aminoglycoside G-418 (Colberre-Garapinet al., 1981, J. Mol. Biol. 150: 1); and hygro, which confers resistanceto hygromycin (Santerre, et al., 1984, Gene 30: 147) genes. Additionalselectable genes have been described, namely trpB, which allows cells toutilize indole in place of tryptophan; hisD, which allows cells toutilize histinol in place of histidine (Hartman & Mulligan, 1988, Proc.Natl. Acad. Sci. USA 85: 8047); and ODC (ornithine decarboxylase) whichconfers resistance to the ornithine decarboxylase inhibitor,2-(difluoromethyl)-DL-ornithine, DFMO (McConlogue L., 1987, In: CurrentCommunications in Molecular Biology, Cold Spring Harbor Laboratory ed.).

Once the leptin protein is expressed by any of the aforementionedsystems, supernatants of the cultured cells or cell lysates may besubjected to various standard methods of protein purification. Forexample, an anti-leptin antibody or Hu-B1.219 protein can be used topurify it by affinity chromatography. Alternatively, leptin may bepurified by ion exchange chromatography or HPLC. Thereafter, the purityof the protein can be confirmed by various methods, including SDS-PAGE,and the protein used immediately or stored frozen for future use. Forcell cultures and in vivo administration, purified leptin must besterilized prior to use.

5.4. Activation of HU-B1.219-Expressing Cells by Leptin

Since various forms of Hu-B1.219, including OB-R, are essentiallyidentical in the extracellular domain, these receptors can bind leptinas a ligand. In order to compare the binding affinity of Hu-B1.219isoforms to leptin, the variant forms are cloned into standardexpression vectors, e.g. CMV promoter expression vectors, andtransfected into COS and BaF3 cells. Surface expression of the receptoris then evaluated by direct binding to an anti-Hu-B1.219 antibody. Inaddition, leptin binding assays can also be performed as described byTartaglia et al. (1995, Cell 83: 1263) using a leptin fusion protein orsoluble leptin conjugated to a radiolabel, a fluorescent dye or anenzyme. The results are compared with mock transfected cells as negativecontrols.

Since the four variants of Hu-B1.219 contain different intracellulardomains, these isoforms can be compared with respect to their signaltransduction capabilities to determine the most active form. Stimulationof most if not all hematopoietic receptors with ligands results in therapid phosphorylation of tyrosines, both on the receptors and on acascade of cellular protein kinases (Heidin, 1995, Cell 80: 213-233;Ihle, 1995, Nature 377: 591-594). Phosphorylation of these moleculesresults in an activation signal being propagated ultimately to thenucleus leading to gene activation. Upon ligand binding to anhematopoietin receptor, some of the first molecules to be activated inthis fashion are the JAK (Janus) family of protein kinases (Ziemiecki etal., 1994, Trends Cell Biol. 4: 207-212). These activated kinases, inturn, phosphorylate members of the STAT family of molecules whicheventually translocate to the nucleus and form active transcriptioncomplexes (Darnell et al., 1994, Science 264: 1415-1421; Zhong et al.,1994, Proc. Natl. Acad. Sci. USA 91: 4806-4810; Hou et al., 1994,Science 265: 1701-1706).

Therefore, cell activation by leptin can be evaluated by studying thepattern of phosphorylation of JAK1-3 following Hu-B1.219 binding. Thiscan be carried out by culturing hematopoietic cells or Hu-B1.219transfectants (1-100×10³), in RPMI 1640 (GIBCO) with gentamicin (100μg/ml), 2 mM glutamine (GIBCO), 10% FCS, and leptin (from 0 to 500 nM)for 10-60 minutes at 37° C. Following the incubation, the cells arewashed in ice-cold PBS and resuspended in lysing buffer (1% TritonX-100, 200 mM NaCl, 10 mM Tris pH 7.5, 2.5 mM p-nitrophenylguanidinobenzoate, 100 μM Na₃VO₄, 1 μM pepstatin, 50 μM3,4-dichloroisocoumarin, 1 mM PMSF, 10 μg/ml leupeptin, 10 μg/mlaprotinin) for 30 minutes at 4° C. followed by removal of nuclei andinsoluble material by centrifugation. To the cell lysate is addedpolyclonal antibodies to JAK1 and JAK2 (Upstate Biotechnology, LakePlacid, N.Y.) or human Tyk2, a human kinase related to the JAK family(Santa Cruz, Calif.) according to manufacturer's recommendation. Thismixture is rotated for 30-60 minutes at 4° C. and then 100 μl of a 10%protein A-sepharose solution (Sigma) is added and rotated for anadditional 30 minutes at 4° C. The precipitate is washed with lysisbuffer and eluted in standard SDS reducing sample buffer and resolved bySDS-PAGE. The proteins are analyzed by Western blots by transferring toImmobilon membranes (Millipore). The membranes are blocked with gelatin(1%) and incubated with anti-phosphotyrosine (4G10, UpstateBiotechnology, Lake Placid, N.Y.) according to the manufacturer'srecommendations. Phosphorylated proteins are detected with ¹²⁵I-labeledprotein A (Amersham).

For the aforementioned experiment, hematopoietic cells can be obtainedfrom human CD34⁺ bone marrow and cord blood. For the murine experiments,three primitive populations can be evaluated, e.g. Ly-6⁺ lineagenegative bone marrow, or the equivalent in fetal liver, or AA4.1⁺ fetalliver.

While tyrosine phosphorylation is an indication of cell activationresulting from ligand-receptor interactions, additional manifestationsof activation at the cellular level can be assayed by using leptin toinduce the proliferation and/or differentiation of hematopoieticprecursors. Cell proliferation can be easily assessed by ³H-thymidineuptake or by visual enumeration of cell numbers. Differentiation ofhematopoietic cells can be assayed by testing the ability of leptin tostimulate the in vitro growth and differentiation of varioushematopoietic colonies. For example, a cell mixture can be isolated fromthe yolk sac, fetal liver or bone marrow, and cultured in standardmethylcellulose colony assays with and without serum supplements, in thepresence of leptin (0.1-500 nM) with or without various other cytokines(Metcalf, 1984, in Clonal Culture of Hematopoietic Cells: Techniques andApplications, Elsevier, N.Y.).

A standard protocol for such an assay involves density gradientcentrifugation of a cell mixture by Ficoll-Hypaque (1.077 gm/cm³) andresuspending about 1×10⁶ viable cells in Iscove's Modified Dulbecco'sMedium (IMDM) with 0.5-15% fetal calf serum (FCS). The cells(1-100×10³/ml) are then mixed with IMDM that contains methylcellulose(1.3%), FCS (0.5-15%), BSA (1%), monothioglycerol (100 μM), gentamicin(50 μg/ml), and leptin (0.1-500 nM). In parallel cultures, additionalcytokines may include: IL-3 (100 pg/ml), steel factor or c-Kit ligand orSCF (10 ng/ml), and EPO (2 U/ml). After mixing, 1 ml of the mixture isdispensed into a 25 mm bacterial grade culture dishes at 37° C. in ahumidified incubator for 5 to 18 days. Using an inverted microscope thenumber and type of hematopoietic colonies are determined. The colonymorphology is used to categorize the various colony types, e.g. BFU-e,CFU-e, CFU-G, CFU-GM, CFU-M, CFU-blast, or CFU-fibroblast (Metcalf,1984, Clonal Culture of Hematopoietic Cells: Techniques andApplications, Elsevier, N.Y.; Freshney, 1994, in Culture ofHematopoietic Cells, Wiley-Liss, Inc., pp. 265-68). Optimalconcentrations of leptin in this assay may increase both the size andthe frequency of these primitive pluripotent colonies.

While leptin may be used alone, it can also be used synergistically withseveral cytokines to promote hematopoietic cell growth including but notlimited to, IL-1, IL-3, IL-6, EPO, steel factor (SCF) and GM-CSF (1ng/ml). In addition, since biologically active leptin is present infetal calf serum (FCS), cultures with 0.5-15% of FCS can be tested. Thespecific activity of leptin in FCS can be inhibited by an anti-leptinantibody or a soluble variant of Hu-B1.219 as a control.

In particular, the effects of leptin may be tested on the primitiveprecursors that form high proliferative potential cells (HPPC) (McNieceand Briddell, 1994, in Culture of Hematopoietic Cells, Wiley-Liss, Inc.,pp. 23-40) and blast colonies (Leary and Ogawa, 1994, in Culture ofHematopoietic Cells, Wiley-Liss Inc., pp. 41-54). In order to evaluatethe effects of leptin on even more primitive cells in these assays,CD34⁺ bone marrow, cord blood, and fetal liver cells can be first sortedby an antibody and tested in the above assays. Since recent evidence hassuggested the existence of a CD34⁻ stem cell, the CD34⁻ Lin⁻ populationmay also be stimulated by leptin.

Furthermore, the effect of leptin on the primitive long term cultureinitiating cells (LTCIC) and on hematopoietic stem cells can beevaluated. LTCIC are precursors that can initiate a long-termhematopoietic culture and are believed to be a function of hematopoieticstem cells (Sutherland and Eaves, 1994, in Culture of HematopoieticCells, Wiley-Liss, Inc., pp. 139-162; Van der Sluijs et al., 1990, Exp.Hematol. 18: 893-896; Traycoff et al., 1994, Exp. Hematol. 22: 215-222).The ability of these culture conditions to expand the hematopoietic stemcell can be confirmed by the competitive repopulation assay (Harrison,1980, Blood 55: 77-81). This assay allows for the quantification ofhematopoietic stem cells.

Additionally, since endothelial cells also express Hu-B1.219, leptin maybe used to stimulate the growth of primary endothelial cells at 0.1-500nM in standard cultures for maintaining primary endothelial cells (Masekand Sweetenham, 1994, Br. J. Haematol. 88: 855-865; Visner et al., 1994,Am. J. Physiol. 267: L406-413; Moyer et al., 1988, In Vitro Cell Dev.Biol. 24: 359-368). Alternatively, leptin may be used to induceendothelial cells to produce cytokines (Broudy et al., 1987, J. Immunol.139: 464-468; Seelentag et al., 1987, EMBO J. 6: 2261-2265).Supernatants of 2-5 day primary endothelial cell cultures or endothelialcell lines cultured in the presence of leptin with or without othercytokines, such as vascular endothelial growth factor (VEGF),platelet-derived growth factor (PDGF), fibroblast growth factor (FGF),transforming growth factor (TGF) and epidermal growth factor (EGF), areharvested and tested as a supplement in the hematopoietic colony assaysabove.

In another aspect of the invention, since receptors often form dimers onthe cell surface, the combination of different Hu-B1.219 isoforms thatgive optimal signal transduction can be measured by growth stimulationand phosphorylation patterns. A Hu-B1.219⁻ growth factor-dependentindicator cell line (e.g. BaF3) can be transfected with variouscombinations of the isoforms using standard CMV expression vectors.Following the demonstration of cell surface expression, leptin (0.1-500nM) is added to the cultures followed by the measurement of growth ratesand phosphorylation patterns by established techniques. Other cell linessuch as TF-1, FD5 and TS1 may also be used.

Because of the expression of isoforms with truncated cytoplasmic tails,it is possible that another protein chain is used in some tissues as thesignaling molecule in association with Hu-B1.219. Such a molecule can bescreened and selected by transfecting pools of cDNA from expressionlibraries of a variety of tissues e.g. fetal liver, CD34⁺ bone marrow,lung, ovary, etc. together with constructs expressing one of thetruncated Hu-B1.219 isoforms into a growth factor-dependent cell line(e.g. BaF3). The ability to grow in the presence of leptin is used as areadout. In particular, the insulin receptor-related receptor (Zhang andRoth, 1992, J. Biol. Chem. 267: 18320), the LIFRα, IL-2Rγ, IL-4Rα(Mosely et al., 1989, Cell 89: 335) and IL-13Rα chains (Hilton et al.,1996, Proc. Natl. Acad. Sci. USA 93: 497) may function as complementarychains for Hu-B1.219 activity. The techniques to identify the uniquecDNA responsible for this complementation are well established in theart.

Additionally, agents that activate Hu-B1.219 in a manner similar toleptin may also be used in place of leptin. These agents include smallmolecules and peptides, and they may be selected in the followingscreening assay. Ten thousand BaF3 and BaF3/Hu-B1.219 (transfectantcells that express the full length Hu-B1.219 isoform) cells will bescreened in microtiter plates for proliferative effects after incubationwith a test agent. Without stimulation these cells die. Any growthpromoting effect seen on the transfected cell line and not with the BaF3host would indicate that the test agent specifically activates theHu-B1.219 receptor or its signaling pathway. This assay is used toscreen small molecules, including peptides, oligonucleotides, andchemical libraries.

5.5. In Vitro Uses of Leptin

In view of the expression of Hu-B1.219 in diverse cell types, leptin maybe used to activate these cells in culture. The activated cells expandin number due to increased proliferation and/or differentiate to becomemore mature cells. In this connection, the optimal effectiveconcentration of leptin for each cell type may be determined byconventional titration experiments in which different amounts of leptinare incubated with the specific cells and their activation levelsmeasured by tyrosine phosphorylation, proliferation or differentiation.

In particular, hematopoietic progenitor cells express Hu-B1.219.Hematopoietic cells may be activated by exposure to leptin in vitrowhich results in their expansion in number prior to their use in vitroand in vivo. Such hematopoietic cells may be obtained from the bonemarrow, the peripheral blood (Demuynck et al., 1995, Ann. Hematol. 71:29-33; Scheding et al., 1994, Stem Cells 12: Suppl. 1: 203-210) and cordblood. In order to selectively enhance the growth of hematopoietic stemcells, the donor cell mixture may be first separated into CD34⁺ cells,followed by leptin stimulation. The expanded cells may be used as donorcells in bone marrow transplantation or as long-term bone marrowcultures (Ponchio et al., 1995, Blood 86: 3314-3321; Testa and Dexter,1991, Curr. Opin. 3: 272-278; Naughton and Naughton, 1991, U.S. Pat. No.5,032,508) for in vitro cytotoxicity testing and the discovery of novelcytokines.

Since Hu-B1.219 is also expressed in some endothelial cells and theirprogenitors, leptin may be used to induce blood vessel formation invitro. In that regard, leptin may be used alone or in combination withVEGF, PDGF, FGF or TGF-α. Blood vessels form by a combination of twoprimary processes. Some blood vessel growth depends on angiogenesis, ina process similar to that associated with pathological conditions. Forinstance, the CNS depends solely on angiogenesis for development of itsvascular supply (Nodew, 1989, Am. Rev. Respir. Dis. 140: 1097-1103;Risau et al., 1988, EMBO J. 7: 959-962). A second process,vasculogenesis, depends on the incorporation of migratory individualendothelial cells (angioblasts) into the developing blood vessel. Leptinmay be used to promote both angiogenesis and vasculogenesis.

In addition, the coding sequence of leptin may be inserted in anexpression vector in accordance with Section 5.3, supra, and transferredinto leptin-nonproducing cell types to result in endogenous expressionof leptin. For example, hematopoietic stem cells are isolated andtransfected with the leptin coding sequence. Thereafter, the cells aretransferred into a bone marrow transplant recipient to cause endogenousproduction of leptin in stimulating hematopoiesis (U.S. Pat. No.5,399,346).

5.6. In Vivo Uses of Leptin

The appropriate dosage and formulation of leptin on human hematopoieticand endothelial development in vivo can be first determined in animalmodels. For example, normal mice may be lethally irradiated orchemically ablated and reconstituted with syngeneic or allogeneic bonemarrow. Since recombinant human leptin has been shown to be active onmouse cells, the rate of donor cell engraftment can be compared betweenleptin-treated and non-treated groups. Because of the Hu-B1.219expression on primitive hematopoietic cells, leptin facilitates thegrowth and recovery of these cells in the recipient. The effect ofleptin on in vivo hematopoietic proliferation and differentiation inthese situations can be evaluated by reconstituting lethally irradiatingmice (900R) with normal bone marrow cells (1-5×10⁶ per mouse). Groups ofanimals are given PBS injections as controls and other groups receiveleptin (0.1-10 mg/kg) at varying intervals. The activity of leptin isassayed by the rapidity of re-normalization and stability of bloodprofiles (e.g. hematocrit, WBC count, differential, etc.).

In addition, neonatal, sublethally irradiated adult normal or SCID micecan be reconstituted with human hematopoietic stem cell isolated frombone marrow, cord blood, or fractions thereof. In these mice, the humanhematopoietic cells engraft and differentiate (McCune et al., 1988,Science 241: 1632-1639; Sandhu et al., 1994, J. Immunol. 152:3806-3813). The effects of leptin can be evaluated by measuring thegrowth rate and extent of differentiation of human cells in theseanimals.

An effective amount of leptin may be administered into a patient who isin need of increased hematopoietic cell function. Such a need may arisefrom various forms of immunodeficiencies (B cell deficiencies, T celldeficiencies and combined deficiencies), myelosuppression, anemias andcancer. In these cases, leptin may be used alone or in combination withother cytokines. Additionally, since certain tumor cells such asleukemic cells express Hu-B1.219, leptin may be used therapeutically tosuppress tumor growth by inducing terminal differentiation of thesecells. Alternatively, leptin may be conjugated to a growth inhibitoryagent such as ricin, diphtheria toxin or a chemotherapeutic drug tospecifically target and destroy tumor cells. Furthermore, antagonists ofleptin, e.g., a modified leptin molecule or a fragment thereof thatbinds to its receptor but does not trigger signal transduction may beused to block the stimulatory effects of leptin in cases where naturallyoccurring leptin stimulates tumor growth in vivo.

Additionally, the leptin coding sequence may be inserted in a viralvector for use in gene therapy (Jolly, 1994, Cancer Gene Therapy, 1:51). In particular, retrovirus, adenovirus, vaccinia virus andadeno-associated viruses are preferred. The leptin codingsequence-carrying virus is injected into a patient to directly supplyleptin by secretion into the bloodstream or to a specific target tissue.

5.6.1. Dosage Determination

The leptin protein, and nucleic acid sequences described herein can beadministered to a patient at therapeutically effective doses to treat orameliorate various hematologic disorders and deficiencies. Atherapeutically effective dose refers to that amount of the proteinsufficient to result in amelioration of symptoms of the disorder, oralternatively, to that amount of a nucleic acid sequence sufficient toexpress a concentration of gene product which results in theamelioration of the disorder.

Toxicity and therapeutic efficacy of leptin can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD₅₀ (the dose lethal to 50% of thepopulation) and the ED₅₀ (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD₅₀/ED₅₀.Purified leptin which exhibits large therapeutic indices is preferred.While leptin preparations that exhibit toxic side effects may be used,care should be taken to design a delivery system that targets suchpreparations to the site of affected tissue in order to minimizepotential damage to normal cells and, thereby, reduce side effects.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofleptin lies preferably within a range of circulating concentrations thatinclude the ED50 with little or no toxicity. The dosage may vary withinthis range depending upon the dosage form employed and the route ofadministration utilized. A dose may be formulated in animal models toachieve a circulating plasma concentration range that includes the IC50(i.e., the concentration of leptin which achieves a half-maximalinhibition of symptoms) as determined in cell culture. Such informationcan be used to more accurately determine useful doses in humans. Levelsin plasma may be measured, for example, by high performance liquidchromatography.

5.6.2. Formulation and Administration

Pharmaceutical compositions for use in accordance with the presentinvention may be formulated in conventional manner using one or morephysiologically acceptable carriers or excipients. The compositions maybe formulated for parenteral administration i.e., intravenous,subcutaneous, intradermal or intramuscular, via, for example, bolusinjection or continuous infusion. Formulations for injection may bepresented in unit dosage form, e.g., in ampoules or in multi-dosecontainers, with an added preservative. The compositions may take suchforms as suspensions, solutions or emulsions in oily or aqueousvehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents. Alternatively, the activeingredient may be in powder form for constitution with a suitablevehicle, e.g., sterile pyrogen-free water, before use. It is preferredthat leptin be introduced into patients via intravenous administrationto directly stimulate blood progenitors.

Leptin may be formulated for administration by inhalation orinsufflation (either through the mouth or the nose) or oral, buccal,parenteral or rectal administration.

For oral administration, the pharmaceutical compositions may take theform of, for example, tablets or capsules prepared by conventional meanswith pharmaceutically acceptable excipients such as binding agents(e.g., pregelatinised maize starch, polyvinylpyrrolidone orhydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystallinecellulose or calcium hydrogen phosphate); lubricants (e.g., magnesiumstearate, talc or silica); disintegrants (e.g., potato starch or sodiumstarch glycollate); or wetting agents (e.g., sodium lauryl sulphate).The tablets may be coated by methods well known in the art. Liquidpreparations for oral administration may take the form of, for example,solutions, syrups or suspensions, or they may be presented as a dryproduct for constitution with water or other suitable vehicle beforeuse. Such liquid preparations may be prepared by conventional means withpharmaceutically acceptable additives such as suspending agents (e.g.,sorbitol syrup, cellulose derivatives or hydrogenated edible fats);emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles(e.g., almond oil, oily esters, ethyl alcohol or fractionated vegetableoils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates orsorbic acid). The preparations may also contain buffer salts, flavoring,coloring and sweetening agents as appropriate.

Preparations for oral administration may be suitably formulated to givecontrolled release of the active compound.

For buccal administration the compositions may take the form of tabletsor lozenges formulated in conventional manner.

For administration by inhalation, the compositions for use according tothe present invention are conveniently delivered in the form of anaerosol spray presentation from pressurized packs or a nebuliser, withthe use of a suitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In the case of a pressurized aerosol the dosage unitmay be determined by providing a valve to deliver a metered amount.Capsules and cartridges of e.g. gelatin for use in an inhaler orinsufflator may be formulated containing a powder mix of the protein anda suitable powder base such as lactose or starch.

The compositions may also be formulated in rectal compositions such assuppositories or retention enemas, e.g., containing conventionalsuppository bases such as cocoa butter or other glycerides.

In addition to the formulations described above, the compositions mayalso be formulated as a depot preparation. Such long acting formulationsmay be administered by implantation (for example subcutaneously orintramuscularly) or by intramuscular injection. Thus, for example, thecompositions may be formulated with suitable polymeric or hydrophobicmaterials (for example as an emulsion in an acceptable oil) or ionexchange resins, or as sparingly soluble derivatives, for example, as asparingly soluble salt.

The compositions may, if desired, be presented in a pack or dispenserdevice which may contain one or more unit dosage forms containing theactive ingredient. The pack may comprise metal or plastic foil, such asa blister pack. The pack or dispenser device may be accompanied byinstructions for administration.

6. EXAMPLE The OB-R is a Variant Form of the Hematopoietin ReceptorDesignated Hu-B1.219

6.1. Materials and Methods

6.1.1. Northern Blot Analysis

In order to study the expression of the Hu-B1.219 gene, Northern blotscontaining RNA obtained from a variety of human tissues (Clontech, PaloAlto, Calif.) were hybridized with a radiolabelled 530 base pair (bp)DNA probe corresponding to nucleotides #578 through 1107 (see FIG.1A-1G). Briefly, the blots were prehybridized at 42° C. for 3-6 hours ina solution containing 5×SSPE, 10× Denhardt's solution, 100 μg/ml freshlydenatured, sheared salmon sperm DNA, 50% formamide (freshly deionized),and 2% SDS. The radiolabelled probe was heat denatured and added to theprehybridization mix and allowed to hybridize at 42° C. for 18-24 hourswith constant shaking. The blots were rinsed in 2×SSC, 0.05% SDS severaltimes at room temperature before being transferred to a wash solutioncontaining 0.1×SSC, 0.1% SDS and agitated at 50° C. for 40 minutes. Theblots were then covered with plastic wrap, mounted on Whatman paper andexposed to x-ray film at −70° C. using an intensifying screen.

6.1.2. Reverse Transcription/Polymerase Chain Reaction (RT/PCR)

Total RNA was isolated using standard laboratory procedures (Sambrook etal., 1989, Molecular Cloning, A Laboratory Manual, Cold Spring HarborLaboratory, NY). Approximately 1 μg of total RNA was reverse transcribedand the cDNA was amplified by PCR (Perkin Elmer, Norwalk, Conn.). ThePCR amplification conditions were the same for Hu-B1.219 and Form 1expression analysis. They were: 94° C. for 30 sec, 60° C. for 30 sec,72° C. for 30 sec for a total of 40 cycles. The amplified products (224bp for Hu-B1.219 and 816 bp for Form 1) were resolved by agarose gelelectrophoresis and visualized by ethidium bromide staining. TheHu-B1.219 amplimers were GGTTTGCATATGGAAGTC (upper)(SEQ ID NO: 13) andCCTGAACCATCCAGTCTCT (lower)(SEQ ID NO: 14). The Form 1 specificamplimers were GACTCATTGTGCAGTGTTCAG (upper)(SEQ ID NO: 15) andTAGTGGAGGGAGGGTCAGCAG (lower)(SEQ ID NO: 16). The upper amplimer wascommonly shared by all 3 forms, whereas the lower amplimer was Form1-specific. The OB-R-specific (Form 4) amplimers were ACATCTTCCCAAAATAGC(upper)(SEQ ID NO: 17) and TGCCTGGGCCTCTATCTC (lower)(SEQ ID NO: 18).

6.2. Results

A number of cDNA clones were isolated from a human fetal liver cDNAlibrary (Clontech, Palo Alto, Calif.), and the DNA sequences of severalof these clones were determined. These clones (designated Hu-B1.219 #4,#33, #34, #1, #36, #8, #55, #60, #3, #57, #62) contained overlappingsequences, which were then compiled into a contiguous nucleotidesequence. Both the nucleotide sequence and the predicted proteinsequence from one such cDNA are shown in FIG. 1A-1G. This cDNA sequencecontains two FN III domains, each containing a “WS box”, which arecharacteristic of genes of the HR family. Thus, this cDNA represents anovel member of the HR gene family, herein referred to as Hu-B1.219(Table 1). Based on the sequence of Hu-B1.219 presented in FIG. 1A-1G,the translation initiation site appears at position #97. The sequenceencodes an open reading frame up to and including nucleotide #2970. Itis believed that the sequence from about nucleotide #2629 to about #2682encodes a transmembrane domain. The complete sequence encodes a proteinof 958 amino acids.

Subsequent amino acid sequence comparison of this molecule with otherpublished protein sequences revealed that it was highly homologous to arecently published human OB-R protein (Tartaglia, 1995, Cell 83:1263-1271). In this connection, the sequence of Hu-B1.219 shown in FIG.1A-1G differs from the published human OB-R sequence only at threenucleotide positions in the extracellular domain, i.e. nucleotideresidues #349, #422 and #764, resulting in amino acids alanine, arginineand arginine, respectively, in Hu-B1.219 protein. The two molecules areidentical in the transmembrane region and a portion of the intracellulardomain up to and including nucleotide #2769, then they diverge atnucleotide #2770 and beyond. TABLE 1 CYTOKINE RECEPTOR GENE FN IIIDOMAIN SIZES (BP) Gene Human Mouse Rat Hu-B1.219(5′) 273 Hu-B1.219(3′)282 IL-2Rβ 291 288 291 IL-2Rγ 273 IL-3Rα 246 252 IL-3RβAic2a 306 and 273IL-3RβAic2b 306 and 282 303 and 276 IL-4R 294 291 IL-5Rα 276 273 IL-6R288 285 gp130 288 291 288 IL-7R 294 IL-9R 321 321 mpl 270 G-CSFR 300 297GM-CSFR 288 CNTFR 282 285 PRLR 288 EPOR 288 285 288 LIFR-1 321 and 297

In addition to the sequence in FIG. 1A-1G referred to as Form 1 ofHu-B1.219 and the variant form reported to be OB-R, other lambda cloneswere discovered that contained different sequences from Form 1 near the3′ end known as Form 2 and Form 3. All three forms contain the identicalsequence up to and including nucleotide #2770, then they diverge atnucleotide #2771 and beyond (FIG. 2). An alignment of the deduced aminoacid sequences of all three forms and the OB-R is shown in FIG. 3A-3F.Two of the originally isolated lambda clones, #36 and #8, contain the 3′end sequences of Form 1 and Form 2, respectively. The different forms ofHu-B1.219 may derive from a common precursor mRNA by an alternativesplicing mechanism. The sequence in this region is consistent with wellknown splice junctions. It is noteworthy that the DNA sequence of Form 1from nucleotide #2768 to the end is 98% identical to a humanretrotransposon sequence that is thought to be derived from a humanendogenous retroviral DNA sequence (Singer, 1982, Cell 28: 433; Weineret al., 1986, Ann. Rev. Biochem. 55: 631; Lower et al., 1993, Proc.Natl. Acad. Sci. USA 90: 4480; Ono et al., 1987, Nucl. Acid Res. 15:8725-8735).

In view of the foregoing, the recently published human OB-R by Tartaglia(1995, Cell 83: 1263) represents a fourth form of Hu-B1.219 because ofits structural similarities to the aforementioned three forms (FIG.3A-3F). Although there are three amino acid substitutions in the OB-R,such differences may have resulted from allelic disparities betweengenetically diverse individuals. It is also possible that the threeforms of the receptor described herein are specific isoforms in fetaland hematopoietic cells, while the OB-R is expressed in the brain. Thedifferences in their intracellular domains may be involved in thedifferent signalling pathways used by these receptor variants indifferent cells.

In order to examine the expression of the variant forms of cDNA, RT/PCRwas performed using several human cell lines. The results in Table 2show that Form 1 was expressed as RNA in K-562 cells and in a humanfetal liver cDNA preparation. Since Hu-B1.219 was cloned from humanfetal liver cDNA library, this served as a positive control. However,with respect to several other human cell lines, Form 1 was not detected,whereas Hu-B1.219 expression was positive. For example, Form 1 was notexpressed in KGla cells, but Form 3 was expressed. Thus, it is possiblethat different forms of Hu-B1.219 are not expressed simultaneously inthe same cells. There may be selective expression of certain forms inparticular cell populations. Additionally, Table 3 shows expression ofHu-B1.219 in cell lines of diverse origins, including hematopoietic,endothelial, central nervous system (CNS), breast and muscle. It isinteresting to note that the variant (Hu-B1.219.4) reported to be OB-Ris also detected in these cells, particularly in hematopoietic celllines, HEL and K562 (Table 3). TABLE 2 RT/PCR ANALYSIS OF COMPARATIVEEXPRESSION OF TWO HU-B1.219 FORMS Cell Lines Hu-B1.219* Form 1Δ Form 3ΔMRC5 (Lung fibroblast) ++ +/− + KG1a (lymphoblast) + − ++ Raji (B celllymphoma) + − + Kit 225/K6 (T cell) +++ − + K562 (myelogenous leukemia)++++ +++ ++++ Human Fetal Liver (positive +++ +++ +++ control)*Analysis by Northern blotsΔAnalysis by RT/PCR

Various human tissue RNA were probed with a radiolabelled Hu-B1.219fragment corresponding to nucleotide numbers from #578 to #1107 asdisclosed in FIG. 1A-1G for Northern blot analyses. Two different sizemRNAs were detected. This result suggests that there may be anotherhomologous gene or there is alternative splicing of a single RNAtranscript. Hu-B1.219 expression was by far the strongest in human fetaltissues, particularly the liver and lung. Trace levels were found inseveral adult tissues. Interestingly, a chronic myelogenous leukemiacell line, K562 (Table 2), was strongly positive for its expression,while some expression was also detected in A549 cells, a lung carcinomacell line (Table 4). A representative northern lot showing theexpression of Hu-B1.219 in several human tissues is presented in FIG. 4.Using more sensitive PCR, Hu-B1.219 was also detected in bone marrow.TABLE 3 EXPRESSION OF Hu-B1.219 IN CELL LINES BY PCR Tissue Type CellLines Hu-B1.219 Hu-B1.219.1 Hu-B1.219.4 Hematopoietic K562 ++++ +++ ++HEL ++++ ++ ++++ Mo7e + +/− − Endothelial HYSE ++++ ++ ++ HYS-VS1 + − +HuVEC ++ + − ECV304 ++ + − CNS U188MG ++ + − SF295 + + +/− U251 ++ + +SNB75 + + + U87MG +++ ++ ++ SNB19 ++ + + SF539 ++ + + Breast DU4475 ++++ ++ MCF-7 + + +/− Muscle 143B ++ + + fetal +++ ++ +++ myoblastHu-B1.219 refers to all isoforms detected.Hu-B1.219.1 refers to Form 1 only.Hu-B1.219.4 refers to the isoform reported to be human OB-R.

TABLE 4 SUMMARY OF NORTHERN BLOT ANALYSIS OF Hu-B1.219 EXPRESSION INHUMAN TISSUES AND CELL LINES Developmental Stage Tissue Type Expressionfetal brain − lung +++ liver +++++ kidney + adult heart ++ brain +/−placenta + lung + liver +++ skeletal muscle + kidney +/− pancreas +spleen +/− thymus +/− prostate ++ testis +/− ovary +++ small intestine++ colon − peripheral blood − leukocytes cancer HL-60 − HeLa − K-562 +++MOLT-4 − Raji − SW480 − A549 + G361 −

Taken together, the data indicates that the Hu-B1.219 represents a newmember of the human hematopoietin receptor family. It was originallycloned from a hematopoietic tissue, fetal liver. It is expressed bycertain fetal tissues, and shares structural homology with severalreceptors which interact with ligands capable of influencinghematopoietic development. In this regard, it shares certain sequencehomology with the IL-6R, IL-4R, G-CSFR, IL-3R beta chain, gp130, IL-12R,and LIFR. It contains two “WS box” motifs with the correct spacing ofconserved amino acids in the FN III domains, an amphipathic sequence inblock 3 of the FN III domains, and alternating hydrophobic and basicamino acids in block 6 of the FN III domains. It also contains conservedcysteines in the cysteine rich regions upstream of the FN III domains.

Despite its structural similarities with receptors expressed byhematopoietic cells, the extracellular domain of Hu-B1.219 is nearlyidentical to that of the human OB-R expressed in the brain. In fact,since three variant forms of Hu-B1.219 have been isolated, which showextensive sequence diversity primarily in their intracellularcytoplasmic domains, OB-R may be considered an additional isoform of thesame receptor. The data presented in Table 3 further confirm that theOB-R is expressed not only in cells of the brain, but also inhematopoietic and endothelial cells. Therefore, since leptin binds toOB-R, it is also a ligand that can trigger Hu-B1.219 activation inhematopoietic and endothelial cells that express these receptorvariants.

7. EXAMPLE Hu-B1.219 is Expressed by Long-Term RepopulatingHematopoietic Progenitor Cells

7.1. Materials and Methods

7.1.1. RNA Extraction and cDNA Synthesis

Total RNA was extracted using the recommended procedure for RNAzolreagent (Biotecx). RNA was added at 1 μg/2 μl of a random hexamer primedRT cDNA synthesis reaction. Mock RT reactions were also performed foreach of the experimental samples. RT reactions were incubated at roomtemperature for 10 minutes, 42° C. for 15 minutes, 99° C. for 5 minutesand a 4° C. hold. All PCR reagents were obtained from Perkin Elmer.

7.1.2. PCR Conditions

The quality of each cDNA and mock cDNA was determined by the relativelevel of amplification of the β-actin gene. The conditions for actinamplification were 94° C. for 30 seconds; 55° C. for 30 seconds; 72° C.for 30 seconds for 27 to 30 cycles followed by a 72° C. extension for 5minutes and a 4° C. hold. The DNA sequence of the β-actin primers were(forward) 5′GTGACGGCCCAGAGCAAGAG-3′ (SEQ ID NO: 19) and (reverse)5′-AGGGGCCGGACTCATCGTACTC-3′ (SEQ ID NO: 20).

PCR amplification conditions for murine homolog of Hu-B1.219 and CD34were 94° C. for 30 seconds; 60° C. for 30 seconds; 72° C. for 30 secondsfor 40 to 45 cycles followed by a 72° C. extension for 5 minutes and a4° C. hold. The primer sequences for Hu-B1.219 were (forward)5′-GGTCAGAAGATGTGGGAAA-31 (SEQ ID NO: 21) and (reverse)5′-GTGCCCAGGAACAATTCTT-3′ (SEQ ID NO: 22). These PCR primers amplifiedboth human and murine sequences. The CD34 primer sequences for humanwere (forward) 5′-CTCTTCTGTCCAGTCACAGACC-3′ (SEQ ID NO: 23), and(reverse) 5′-GAATAGCTCTGGTGGCTTGC-3′ (SEQ ID NO: 24). The CD34 primersequences for murine were (forward) 5′-CTACCACGGAGACTTCTACAC-3′ (SEQ IDNO: 25) and (reverse) 5′-TGGATCCCCAGCTTTCTCAA-3′ (SEQ ID NO: 26). ThePCR products ere analyzed on a 1.5% TAE agarose gel containing 0.5 μgthidium bromide/ml of buffer.

7.2. Results

Section 6.2, supra, demonstrates that Hu-B1.219 is expressed inhematopoietic and endothelial cells. In this connection, its expressionin fetal liver is consistent with the high hematopoietic activities inthe fetal liver, and its expression in fetal lung is consistent with thehigh level of endothelial development in fetal lung. To more preciselydetermine the expression of Hu-B1.219 in different populations ofhematopoietic cells, human bone marrow cells were highly enriched forhematopoietic stem cells by cell sorting based on their CD34 expression(Collins et al., 1994, Stem Cells 12: 577-585; Berenson, 1993, J.Hematother. 2: 347-349; Civin and Gore, 1993, J. Hematother. 2:137-144). When RNA extracted from human CD34⁺ and CD34⁻ bone marrowfractions were reacted with Hu-B1.219 primers in PCR, Hu-B1.219 messagewas detected in both fractions (FIG. 5A). The fact that only the CD34⁺fraction expressed CD34 message in PCR demonstrates the purity of thesorted population (FIG. 5B). Since the CD34⁻ fraction contained severalcell types, the detected Hu-B1.219 message might have been produced byendothelial cells. Alternatively, Hu-B1.219 may be expressed by a CD34⁻hematopoietic stem cell.

The expression of Fc receptors (FcR) and AA4.1 antigen in murine fetalliver cells has been used to define distinct fetal liver precursor cellsubpopulations (Carlsson et al., 1995, Eur. J. Immunol. 25: 2308-2317;Jordan et al., 1995, Exp. Hematol. 23: 1011-1015; Trevisan and Iscove,1995, J. Exp. Med. 181: 93-103). Sorting murine fetal liver cells (day12) based on the expression of these two markers has resulted in theisolation of a small (2-4%) subpopulation of AA4.1⁺ and FcR⁻ cells thatare highly enriched for primitive hematopoietic stem cells. Animalrepopulating experiments have shown that fetal liver cells with thisphenotype contain long-term repopulating potential upon adoptivetransfer into recipients with destroyed lympho-hematopoietic system.Therefore, fetal liver cells were sorted into various fractions based onexpression of AA4.1 and FcR, and primers designed from Hu-B1.219 thatwould amplify murine Hu-B1.219 message were used to detect itsexpression. FIGS. 6A and 6B shows that the highly enriched AA4.1⁺/FCR⁻subpopulation was the only population at this stage that expressedHu-B1.219, whereas CD34 mRNA was detected in all fractions tested. Thisresult indicates that while CD34 has been used as a marker ofhematopoietic progenitor cells, Hu-B1.219 marks with greater specificitythe long-term repopulating cells within the CD34⁺ fraction. In addition,Hu-B1.219 expression was also observed in the subpopulation of murineadult bone marrow sorted for Ly-6 expression but negative for maturelineage markers. This fractionation procedure is a well establishedtechnique for isolating a highly purified population of hematopoieticstem cells from bone marrow (Li et al., 1992, J. Exp. Med. 175:1443-1447; Spangrude and Brooks, 1993, Blood 82: 3327-3332; Szilvassyand Cory, 1993, Blood 81: 2310-2320). Furthermore, murine fetal livercells enriched for long-term repopulating cells by a recently developedmethod utilizing expression of the Mac-1 marker also expressed Hu-B1.219(Morrison et al., 1995, Proc. Natl. Acad. Sci. USA 92: 10302-10306).Taken collectively, the aforementioned mouse and human studies indicatethat Hu-B1.219 is a marker of a subpopulation of early progenitor cellswithin the CD34⁺ fraction. In particular, Hu-B1.219 expression inlong-term repopulating cells allows its use as a marker for theirisolation. In this regard, an antibody specific for Hu-B1.219 or leptinmay be used alone or in combination with a progenitor cell-specificantibody such as anti-CD34. For example, human bone marrow cells can beseparated first into CD34⁺ cells followed by Hu-B1.219 sorting to obtainsuch progenitor cells. In addition, since Hu-B1.219 expression isdetected in a CD34⁻ subpopulation, it can also be used as a marker toisolate a CD34⁻ stem cell.

Additionally, early embryonic yolk sac cells have been described topossess the potential of giving rise to both hematopoietic andendothelial cells (Wagner and Antczak, 1995, WO95/02038). In the yolksac, endothelial cells also produce the microenvironment forhematopoietic differentiation and proliferation. It is important to notethat yolk sac cells with endothelial potential (Wei et al., 1995, StemCells 13: 541-547) have also been shown to express Hu-B1.219. Therefore,Hu-B1.219 may also be used as a marker for endothelial progenitor cells.

8. EXAMPLE Recombinant Leptin Stimulates Bone Marrow Colony Formation

8.1. Materials and Methods

8.1.1. Production of Recombinant Leptin

Total RNA was isolated, using RNAzol method, from brown adipose tissuefrom C57B mice. RT-PCR was performed using the Boehringer Mannheim “HighFidelity PCR System.” PCR primers: murine leptin U462GGAATTCCATATGGTGCCTATCCAGAA (SEQ ID NO: 27) and L462GCGGATCCTCAGCATTCAGGGCTAA (SEQ ID NO: 28) were designed based on Genbanksequence U18812 (Zhang et al., 1995, NATURE 372: 425-432). The PCRproduct was purified using the Promega Wizard column. The PCR fragmentwas cut with NdeI and BamHI and cloned into NdeI-BamHI cleaved pET15bvector (Novagen). Clones were obtained first in E. coli strain DH10B(GibcoBRL), then transferred to the BL21 (DE3) (Novagen) host forproduction. Two splicing variants of mOB ere identified. One that wasmissing a single glutamine amino acid at residue #49 from the iniatorMet and one that had a glutamine at that position. Proteins were madefrom both clones. Murine leptin was made in E. coli as a fusion proteinwith poly histidine on the amino terminus. Recombinant murine leptin wasproduced as insoluble inclusion bodies and purified as described inSambrook et al., supra. The insoluble material was denatured and“refolded” to reconstitute biologically active leptin. Inclusion bodieswere dissolved in 8M Urea plus 100 mM DTT, diluted {fraction (1/100)}into a “refolding reaction buffer” (100 mM Tris pH8.3, 100 mM (NH₄)₂SO₄,100 μM Triton X100, 2 mM reduced glutathione, 0.4 mM oxidizedglutathione) and incubated at 4° C. for 3 to 5 days. The refolded leptinwas recovered by adding the histidine binding resin (Novagen) for 1 hr.The resin was recovered by centrifugation, rinsed with Novagen “bindingbuffer”, and the Novagen “wash buffer”. The leptin was eluted from theresin with Novagen “elution buffer” plus 1M imidazole. The final stepwas dialysis in PBS.

8.1.2. Methylcellulose Colony Forming Assays

Adult bone marrow cells were isolated from normal C5-7BL/6 female mice,ob/ob mice and db/db mice. About 1×10⁶ viable cells were suspended inIscove's Modified Dulbecco's Medium (IMDM) with either 0.5 to 15% fetalcalf serum (FCS). The cells (1-100×10³/ml) were then mixed with IMDMthat contained methylcellulose (1.3%) (Sawyer-Biddle, NY, N.Y.), FCS(4%), BSA (1%), monothioglycerol (100 μM), gentamicin (50 μg/ml),purified recombinant leptin (1 ng/ml), IL-3 (100 pg/ml), GM-CSF (1ng/ml) and EPO (2 U/ml). After mixing, 1 ml of the mixture was dispensedinto a 25 mm bacterial grade culture dishes at 37° C. in a humidifiedincubator for 3 days. Using an inverted microscope, CFU-e, BFU-e andGEMM ≧4 cells ere counted (Metcalf, 1984, Clonal Culture ofHematopoietic Cells: Techniques and Applications, Elsevier, N.Y.;Freshney, 1994, in Culture of Hematopoietic Cells, Wiley-Liss, Inc., p.265-68).

8.2. Results

Recombinant leptin was tested for its ability to stimulate bone marrowcells to form hematopoietic colonies in ethylcellulose assays. Whennormal mouse bone marrow cells were incubated with leptin in thepresence of IL-3, GM-CSF and EPO, a two-fold increase in the number ofCFU-e was observed as compared to the cells stimulated with mediumcontaining the cytokines except leptin (FIG. 7). Similarly, leptincaused an increased number of CFU-e from ob/ob mouse bone marrow. Incontrast, leptin did not stimulate CFU-e from db/db mouse bone marrow,an observation consistent with the belief of an aberrant OB-R expressedby such animals (Chen et al., 1996 Cell 84: 491-495).

Additionally, leptin also stimulated other types of colonies from normalmouse bone marrow. For example, BFU-e was induced by leptin incubation,indicating that leptin acts on early progenitor cells of the erythroidlineage (FIG. 8). Furthermore, leptin also stimulated the formation ofearly CFU-GM colonies, indicating its effects on granulocyte, erythroid,macrophage and megakaryocyte lineages. When freshly isolated murine yolksac cells were incubated with leptin alone, cellular proliferation anderythroid development were also induced. In conclusion, theHu-B1.219/OB-R is not only expressed in early hematopoietic progenitorcells, it renders these cells responsive to leptin stimulation resultingin cellular proliferation and differentiation into cell types of diversehematopoietic lineages. Thus, leptin is a growth and differentiationfactor of hematopoietic progenitor cells.

9. Deposit of Microorganisms

The following organisms were deposited with the American Type CultureCollection (ATCC), 12301 Parklawn Drive, Rockville, Md. 20852. StrainDesignation Accession No. Hu-B1.219, #1 75885 Hu-B1.219, #4 75886Hu-B1.219, #8 75887 Hu-B1.219, #33 75888 Hu-B1.219, #34 75889 Hu-B1.219,#36 75890 Hu-B1.219, #55 75971 Hu-B1.219, #60 75973 Hu-B1.219, #3 75970Hu-B1.219, #57 75972 Hu-B1.219, #62 75974

The use in the present application of the computer readable SequenceListing submitted Mar. 27, 1998 in U.S. patent application Ser. No.08/618,957 is hereby requested. The computer readable Sequence Listingon file for U.S. patent application Ser. No. 08/618,957 is identical tothe paper copy (29 pages) submitted with the present application.

The present invention is not to be limited in scope by the exemplifiedembodiments, which are intended as illustrations of individual aspectsof the invention. Indeed, various modifications for the invention inaddition to those shown and described herein will become apparent tothose skilled in the art from the foregoing description and accompanyingdrawings. Such modifications are intended to fall within the scope ofthe appended claims.

All publications cited herein are incorporated by reference in theirentirety.

1-26. (canceled)
 27. A method for identifying hematopoietic progenitorcells in a cell mixture, comprising contacting the cell mixture with anagent that binds to Hu-B1.219 protein and selecting the cells bound tothe agent.
 28. The method of claim 27 in which the agent is an antibodyor a fragment thereof.
 29. The method of claim 27 in which the agent isleptin or a fragment thereof.
 30. A method for detecting hematopoieticprogenitor cells in a cell mixture, comprising: (a) contacting an RNAextracted from the cell mixture with a nucleic acid probe capable ofspecifically hybridizing to at least a portion of a coding strand of acDNA of SEQ ID NO: 1; and (b) detecting hybridization between the probeand the RNA.
 31. A method for identifying hematopoietic progenitor cellsin a tissue, comprising: (a) contacting a tissue with a nucleic acidprobe capable of specifically hybridizing to at least a portion of acoding strand of a cDNA of SEQ ID NO: 1 (b) detecting hybridizationbetween the probe and the RNA of the tissue.
 32. A method for detectingcancer, comprising: (a) contacting a bodily fluid with a specificbinding agent for Hu-B1.219 protein; and (b) detecting the presenceHu-B1.219 in the fluid.